There are numerous instances in scientific and technical endeavors when one desires to observe and measure fluorescent signals. As one example, in the field of biotechnology, it has become common to generate a many-fold increase in a material, such as DNA, RNA or other biologically or medically interesting material, in order to perform tests, obtain diagnoses, do other medically useful procedures, or identify a source of the material for forensic purposes, when only a minute amount of the biologically or medically interesting material is obtained or is available. The amount of material present can be quantified by observing an optical signal, such as a fluorescent signal, from the reaction product itself, or from a substance that chemically combines with the reaction product of interest. By way of example, this is described in many United States patents, including U.S. Pat. Nos. 7,282,579, 7,282,567, 7,282,563, 7,282,557, 7,282,477, 7,282,366, 7,282,360, 7,282,350, 7,282,343, 7,282,335, 7,282,207, 5,869,243, 5,221,610, and 5,077,192, the disclosures of all of which are incorporated herein by reference.
As a broad introduction to these kinds of activities, one can cite the discussion presented by Mullis et al. in U.S. Pat. No. 5,656,493, issued on Aug. 12, 1997, the disclosure of which is incorporated herein by reference. Mullis describes the Polymerase Chain Reaction (PCR) method as an example of a method of increasing the available amount (or “amplifying”) material such as DNA.
There are methods for producing nucleic acid sequences in large amounts from small amounts of an existing sequence. Such methods involve cloning of a nucleic acid sequence in an appropriate host system, and culturing the host, wherein the vector in which the nucleic acid sequence has been inserted is replicated, resulting in copies of the vector and hence the Sequence. See T. Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 390-401 (1982); and U.S. Pat. Nos. 4,416,988 and 4,403,036. The original sequence can also be organically synthesized before insertion in a vector. See U.S. Pat. No. 4,293,652.
A method, described by Saiki et al., Science, 230, 1530-1534 (1985), has been devised for amplifying one or more specific nucleic acid sequences or a mixture thereof using primers, nucleotide triphosphates, and an agent for polymerization, such as DNA polymerase. The extension product of one primer, when hybridized to the other, becomes a template for the production of the desired specific nucleic acid sequence, and vice versa. The process is repeated as often as necessary to produce the desired amount of the sequence.
This method is especially useful for performing clinical tests on the DNA or RNA from a fetus or other donor where large amounts of the DNA or RNA are not readily available and more DNA or RNA must be manufactured to have a sufficient amount to perform tests. The presence of diseases which have unique DNA or RNA signatures can be detected by amplifying a nucleic acid sample from a patient and using various probe procedures to assay for the presence of the nucleic acid sequence being detected in the test. Such test might be prenatal diagnosis of sickle cell anemia, as described by Saiki et al., supra, where the amplification of specific B-globin target sequences in genomic DNA resulted in the exponential increase (220,000 times) of target DNA copies, increasing sensitivity and speed while reducing the complexity of diagnosis. Another test is the diagnosis of the AIDS virus, which is thought to alter the nucleic acid sequence of its victims.
Five patent applications which describe the amplification process, PCR, are U.S. patent application Ser. No. 818,127, filed Jan. 10, 1986, now abandoned, U.S. Ser. No. 716,982, filed Mar. 28, 1985, now U.S. Pat. No. 4,683,194, U.S. Ser. No. 791,308, filed Oct. 25, 1985, now U.S. Pat. No. 4,683,202, U.S. Ser. No. 828,144, filed Feb. 7, 1986, now U.S. Pat. No. 4,683,195, and U.S. Ser. No. 839,331, filed Mar. 13, 1986, now abandoned, the disclosures of all of which are incorporated herein by reference.
The amplification method, PCR, bears some similarity to the molecular cloning methods described above, but does not involve propagation of a host organism, avoiding the hazards and inconvenience therein involved. In addition, the amplification method does not require synthesis of nucleic acid sequences unrelated to the desired sequence, and thereby obviates the need for extensive purification of the product from a complicated biological mixture. Finally, the amplification is more efficient than the alternative methods for producing large amounts of nucleic acid sequences from a target sequence and for producing such sequences in a comparatively short period of time.
At first, the amplification procedure, PCR, described above was carried out by hand in the laboratories. The manual process involves a great deal of repetitive liquid handling steps and incubations at controlled temperatures. This is not only time-consuming and tedious, but it is also subject to error caused by human operator attention span drift. Such errors could result in a misdiagnosis of a genetic birth defect and an unnecessary abortion or the lack of an abortion where a birth defect exists. Further, such errors could result in misdiagnosis of sickle cell anemia or other genetic disorders.
Further, certain nucleic acids amplify more efficiently than others, so some nucleic acid sequence amplifications require more amplification cycles than others because the cost of laboratory labor can be high, and the risks to which a laboratory is subjected are high in case of error in erroneously performing amplification, there has arisen a need for a system which can automate the amplification process.
Mullis then described a system for use in performing an automated amplification process.
The amplification process, PCR, maybe conducted continuously. In one embodiment of an automated process, the reaction may be cycled through a denaturing region, a reagent addition region, and a reaction region. In another embodiment, the enzyme used for the synthesis of primer extension products can be immobilized in a column. The other reaction components can be continuously circulated by a pump through the column and a heating coil in series; thus the nucleic acids produced can be repeatedly denatured without inactivating the enzyme.
One embodiment of a machine for automating the amplification process utilizes a liquid handling system under computer control to make liquid transfers of enzyme stored at a controlled temperature in a first receptacle into a second receptacle whose temperature is controlled by the computer to conform to a certain incubation profile. The second receptacle stores the nucleic acid sequence to be amplified plus certain reagents. The computer includes a user interface through which a user can enter process parameters which control the characteristics of the various steps in the sequence such as the times and temperatures of incubation, the amount of enzyme to transfer on each cycle into the second receptacle from the first receptacle, as well as the number of cycles through the amplification sequence that the user desires the machine to perform. The first and second receptacles may be controlled in temperature by use of three circulating fluid reservoirs and solenoid operated valves. Of course, any other method for controlling the temperatures of the receptacles will also work for purposes of the invention, and the invention is not limited to the use of heated and chilled circulating fluids. These solenoid operated valves are coupled to the computer such that the proper temperature fluid can be directed through the supporting structure for the first and second receptacles at the proper times in the PCR sequence under computer control. The first receptacle, which stores enzyme to be added to the reaction well of the second receptacle, is kept at a constant temperature. The second receptacle, which is where the PCR reaction occurs, is switched under computer control between two temperatures by the transmission of a control signal to the solenoid operated valves at the proper time in the sequence to gate either the hot fluid or the cold fluid through the support structure of the second receptacle.
While the above-described machine increases the amount of nucleic acid sequence which can be amplified per unit of labor, thereby decreasing the possibility of error, it involves liquid handling, where reagents must be continuously transferred at various cycles. There is a need also for a machine which not only automates the amplification process, but also makes it faster and more convenient. This can be accomplished using an enzyme which is thermostable, i.e., will not break down when subjected to denaturing temperatures.
A second embodiment of the invention utilizes a temperature-cycling instrument for implementing the amplification process when a thermostable enzyme is employed. The use of a thermostable enzyme avoids the need for liquid transferring of the enzyme, which is necessitated when the enzyme is unstable in the presence of heat. As used herein to describe enzymes, “thermostable” means stable at temperatures above 90 degree C. and “heat-stable” means stable at temperatures 65 degree-90 degree C.
In U.S. Pat. No. 6,814,934, issued Nov. 9, 2004, which is incorporated herein by reference, Higuchi describes methods of monitoring the amplification of nucleic acids. The sensitivity and specificity of nucleic acid detection methods was greatly improved by the invention of the polymerase chain reaction (PCR). PCR is a process for amplifying nucleic acids and involves the use of two oligonucleotide primers, an agent for polymerization, a target nucleic acid template, and successive cycles of denaturation of nucleic acid and annealing and extension of the primers to produce a large number of copies of a particular nucleic acid segment. With this method, segments of single copy genomic DNA can be amplified more than 10 million fold with very high specificity and fidelity. PCR methods are disclosed in U.S. Pat. No. 4,683,202, which is incorporated herein by reference. PCR and other methods of amplifying biologically or medically interesting material are well known and will not be further discussed herein.
There is a need for an improved apparatus and method for collecting optical signals such as fluorescent signals, so that one can obtain results in shorter times, using less material, and from less concentrated reaction media.